Epithelial cells are polarized cells that target proteins to both structural and functional compartments within the cell. Specific signaling molecules must also localize to these compartments to mediate ligand-specific cellular responses. At present, little is known about the compartmentalization of signaling molecules such as G proteins. In this proposal, we hypothesize that the localization of signaling molecules into discrete mircodomains contributes to the regulation of a unique epithelial cell function, the tight junction barrier. This application proposes to extend preliminary data obtained in the sponsor's laboratory showing a direct interaction between Galpha12 and the tight junction protein ZO-1. Preliminary results in MDCK cell culture systems suggest that activated Galpha12 disturbs barrier function, and this effect may involve signaling through Src tyrosine kinases. Based upon these findings, we aim to (1) define the interacting domains between Galpha12 and ZO-1, and (2) characterize the Galpha12/Src signaling pathway regulating the tight junction in MDCK cells. We hypothesize that the ZO-1 SH3 domain interacts with a potential SH3 binding region found in the N-terminus of Galpha12. In addition, we will determine if Src is associated with the Galpha12/ZO-1 complex and look for direct evidence of increased Src activation. These studies will utilize molecular cloning, protein expression (in-vitro translation and glutathione-S- transferase (GST) fusion proteins), immunoprecipitations, and binding assays as well as MDCK cell culture systems and enzymatic assays. Such experiments will yield insight into the regulation of compartmentalized cellular function and could provide the basis for development of novel therapies involving the epithelial cell tight junction. She has presented abstracts at clinical meetings. In July of this year she will be entering a Fellowship program in Nephrology at Brigham and Women's Hospital where the first three years are devoted to research and the final year to clinical training. Her letters of recommendation are outstanding with high praise for her focus, dedication, her scholarly spirit and her compassion and competence as a clinician. She has been instrumental in developing information technology for her current residency program. Her referees speak strongly of her dedication to cell biology research. The perseverance with which Dr. Hunt has maintained her "finger in the research pie" despite the rigors of Medical school and residency training underscore her commitment to research.